The GAPDH-specific docking sites on δPKC are masked when the δPKC itself is inactive. When the conformational changes occur in δPKC during its activation, GAPDH-specific docking sites get exposed.
The researchers assumed that there must be a pseudoGAPDH-docking site (ψGAPDH) analogous to pseudosubstrate concept. And it is the pseudoGAPDH-docking site which masks the GAPDH-docking site in an inactive state. The ψGAPDH site of the δPKC is the one that mimics the GAPDH binding site of the δPKC when the enzyme is inactive.
In the study, researchers developed a peptide that corresponds to the ψGAPDH and they named that peptide as a ψGAPDH peptide. With the help of ψGAPDH peptide, they identified phosphorylation as a novel switch that controls the oligomeric state of the GAPDH and mediates its function as a metabolic enzyme as well as a mediator of the mitochondrial elimination.
Interaction of GAPDH and δPKC
To determine the level of interaction of glyceraldehyde-3-phosphate dehydrogenase with the δPKC in the presence of ψGAPDH peptide, researchers incubated 200 ng of recombinant δPKC with or without 1 µM of the ψGAPDH peptide for 10 minutes at 4 ºC. After 10 minutes of incubation, 1 µM of ψGAPDH was added followed by another 20 minutes of incubation at 37 ºC.
After incubation, GAPDH was immunoprecipitated using GAPDH specific antibody and δPKC-GAPDH binding was determined using δPKC specific antibody followed by Western blot analysis using HRP-conjugated secondary antibody.
GAPDH phosphorylation by δPKC
To determine the level of phosphorylation of the GAPDH in the presence of ψGAPDH peptide, researchers incubated 200 ng of recombinant δPKC protein with or without 1 µM ψGAPDH for 10 minutes at 4 ºC and then they added 200 ng of recombinant GST-GAPDH followed by 20 minutes of incubation at 37 ºC. The 200 ng of recombinant GST-GAPDH was prepared in a 40 µl kinase buffer containing; 20 mM Tris-HCl, 20 mM MgCl2, 1 µM DTT, 25 µM ATP and 1mM CaCl2 along with PKC activators, 1.25 µg phosphatidylserine and 0.04 µg 1,2 dioleoyl sn-glycerol.
The reaction was stopped by adding Laemmli buffer containing 5 % SDS and the samples were loaded into the 10 % SDS-PAGE and the level of phosphorylated GAPDH protein were determined by using anti-phospho-threonine and anti-phospho-serine PKC substrate antibodies and then with HRP-conjugated secondary antibody using Western blotting.
Mitochondrial content and the effect of siRNA treatment
The researchers carried out cell viability assay of the mice cardiac H9C2 cells and measured the mitochondrial content and the effect of siRNA treatment. They grew the Cardiac H9C2 cells with or without ψGAPDH peptide and after 15 minutes cells were treated with H2O2 followed by incubation. In another set of culture, these cells were transfected with GAPDH or siRNA and after 48 hours of incubation. After that, the cells were treated with 1 mM H2O2 for 1 hour.
After that, researchers isolated the heart mitochondria using type I protease and washed the mitochondrial pellet with the help of isolation buffer and isolated the mitochondrial content. Protein content was measured using Bradford protein assay. PDK phosphorylation was determined using 2D-PAGE and the gel was Western blotted using anti-PDK2, anti-phospho-threonine and anti-phospho-serine PK as substrates.
Phosphorylation of other signaling proteins such as HSP27, MARCKS, STAT and Troponin I were determined using one-dimensional SDS-PAGE and the gel was Western blotted using anti-phospho-HSP27 and anti-HSP27, anti-phospho-MARCKS and anti-MARCKS, anti-phospho-STAT and anti-STAT, anti-phospho-Troponin I and anti-Trop antibodies respectively. The level of GAPDH phosphorylation was determined using anti-phospho-serine PKC substrate and anti-GAPDH. The level of phosphorylation was determined by subtracting with normalized to the total substrate.
Substrate-specific docking sites are prevalent in serine/threonine kinases such as JNKs, CDKs, and peptides derived from such docking sequences can be used as inhibitors to inhibit the phosphorylation of the corresponding substrates.
Here researchers used a simple approach to study the interaction site between the glyceraldehyde-3-phoaphate dehydrogenase and δPKC and showed that sequence derived from the docking site is an inhibitor of the GAPDH-docking site of the δPKC.
ψGAPDH peptide is a selective inhibitor that inhibits the δPKC-mediated phosphorylation of the GAPDH. Glyceraldehyde-3-phosphate dehydrogenase inhibitor identified by these researchers elucidated the role of the GAPDH tetramerization for the glycolysis as well as mitochondrial elimination via mitophagy.
Other non-catalytic roles
Other roles of the glyceraldehyde-3-phosphate dehydrogenase include regulation of the gene expression. Its translocation to the nucleus is associated with the regulation of gene expression responsible for the apoptosis and blocking the translocation can prevent the cytotoxicity.
GAPDH is also involved in the DNA repair, tRNA export and membrane fusion and transport and many more human diseases. As, for example, increased nuclear translocation of GAPDH is associated with the increased neuronal apoptosis in Parkinson’s disease.